Methods for increasing ApoE levels for the treatment of neurodegenerative disease

ABSTRACT

Disclosed herein is a method for reducing neurodegenerative disease in patients by administration of a therapeutically-effective amount of a compound which can increase ApoE levels.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit from provisional application60/059,908 filed on Sep. 24, 1997.

BACKGROUND OF THE INVENTION

[0002] This invention relates to methods for the treatment ofneurodegenerative disease.

[0003] Neurodegenerative diseases include Alzheimer's disease (AD)Creutzfeldt-Jakob disease, Huntington's disease, Lewy body disease,Pick's disease, Parkinson's disease, amyotrophic lateral sclerosis(ALS), neurofibromatosis, and diseases without a necessary geneticcomponent such as brain injury, stroke, and multiple infarct dementia.Most of these diseases are typified by onset during the middle adultyears and lead to rapid degeneration of specific subsets of neuronswithin the neural system, ultimately resulting in premature death. Thereare no known cures and few therapies that slow the progression of thesediseases.

[0004] Alzheimer's disease (AD) is one of the most clinically importantof the neurodegenerative diseases due to the high frequency ofoccurrence within the population and the fatal course of the disease.Two forms of the disease exist: presenile dementia, in which thesymptoms emerge during middle age, and senile dementia which occurs inthe elderly. Both forms of the disease appear to have the samepathology. A clear genetic predisposition has been found for preseniledementia. Familial autosomal dominant cases have been reported and themajority of individuals with trisomy 21 (Down's syndrome) developpresenile dementia after the age of 40. The genetic loci associated withfamilial Alzheimer's disease map to chromosomes 14, 19, and 21, withmore than one locus on 21.

[0005] Pick's disease (lobar sclerosis) is expressed clinically asdementia that is essentially indistinguishable from that of Alzheimer'sdisease. The disease becomes symptomatic in middle adulthood andprogresses relentlessly over a period of 3 to 5 years. Some of Pick'sdisease cases cluster in families, but the case distributions do notconform to a strictly hereditary pattern. Women are affected by thedisease more often than men.

[0006] The brain in Pick's disease is atrophic. Unlike Alzheimer'sdisease, the atrophy is typically localized in a frontal or a temporallobe, and it may attain extreme proportions. Histologically, theinvolved cortex is markedly depleted of neurons, and their absence isaccentuated by a marked astrogliosis. Many residual neurons have a“ballooned” cytoplasm, and in some, there is one or more faintlyeosinophilic inclusions, termed Pick bodies, whose presence is confirmedby their intense argentophilia. These bodies are formed of denselyaggregated neurofilaments.

[0007] Creutzfeldt-Jakob disease was named after the two doctors fromAustria who in 1920 separately described a total of six patients withpeculiar neurological illnesses. Although the illnesses were not verysimilar, the post mortem brain tissue appeared similar when viewed underthe microscope. Many of the normal brain nerve cells were dead and thetissue had numerous tiny holes, resembling a sponge, hence, theexpression “spongiform encephalopathy” is also used to describe thedisease.

[0008] Creutzfeldt-Jakob disease (CJD) occurs worldwide. There isapproximately one new case per two million people per year with the ageof onset occurring between the ages of 55 and 80. CJD can affect morethan one member of a family but this is rare.

[0009] Huntington's disease is a progressive disease which is alwaystransmitted as an autosomal dominant trait mapping to a single locus onchromosome 4. Individuals are generally asymptomatic until the middleadult years, although some patients show symptoms as early as age 15.Once symptoms appear, the disease is characterized by choreoathetoticmovements and progressive dementia until death 15-20 years after the ageof onset.

[0010] Autopsies of Huntington's disease patients reveal progressiveatrophy of the caudate nucleus and basal ganglia. Atrophy of the caudatenucleus and the putamen is seen microscopically where there is anexcessive loss of neural tissue.

[0011] Although some of the characteristic mental depression and motorsymptoms associated with Huntington's may be suppressed using tricyclicantidepressants and dopamine receptor antagonists, respectively, notherapy exists for slowing or preventing the underlying disease process

[0012] Parkinson's disease is a common neurodegenerative disorder whichfirst appears in mid to late life. Familial and sporadic cases occur,although familial cases account for only 1-2 percent of the observedcases. The neurological changes which cause this disease are somewhatvariable and not filly understood. Patients frequently have nerve cellloss with reactive gliosis and Lewy bodies in the substantia nigra andlocus coeruleus of the brain stem. Similar changes are observed in thenucleus basalis of Meynert. As a class, the nigrostriatal dopaminergicneurons seem to be most affected.

[0013] The disorder generally develops asymmetrically with tremors inone hand or leg and progresses into symmetrical loss of voluntarymovement. Eventually, the patient becomes incapacitated by rigidity andtremors. In the advanced stages the disease is frequently accompanied bydementia.

[0014] Diagnosis of both familial and sporadic cases of Parkinson'sdisease can only be made after the onset of the disease symptoms.Anticholinergic compounds, such as propranolol, primidone, and levodopa,are frequently administered to modify neural transmissions and therebysuppress the symptoms of the disease, though there is no known therapywhich halts or slows the underlying progression.

[0015] Lewy body disease is a preferred term which describes severalcommon disorders causing dementia. In many hospitals this is the secondmost common cause of dementia following Alzheimer's disease. The namefor the disease comes from the presence of abnormal lumps called Lewybodies which develop inside nerve cells.

[0016] The primary clinical symptom of this condition is the developmentof dementia, although patients may also show Parkinsonian symptoms andexperience hallucinations.

[0017] Autopsy of Lewy body disease patients reveals degeneration of thesubstantia nigra, as would be seen in Parkinson's disease. In Lewy bodydementia and Parkinson's disease, the melanin containing nerve cells ofthe substantia nigra die and the tissue thus appears abnormally pale incomparison to normal tissue.

[0018] The cause of Lewy body disease is uncertain. However, anassociation with a higher apoE4 allele frequency has been noted.

[0019] Neurofibromatosis actually refers to at least two related butquite distinct diseases.

[0020] Neurofibromatosis 1 (NF1, peripheral neurofibromatosis, vonRecklinghausen disease) is characterized by multiple neurofibromas, cafeau lait macules, and Lisch nodules of the iris. Some patients with NF1also show learning disabilities, macrocephaly, bony abnormalities, and ahigher frequency of neural malignancies.

[0021] Neurofibromatosis 2 (NF2, central neurofibromatosis) ischaracterized by the appearance of bilateral vestibular schwannomas. Themajority of NF2 patients develop schwannomas of cranial or peripheralnerves while a minority develop other tumors, such as meningiomas andependymomas.

[0022] Both NF1 and NF2 are autosomal dominant disorders with nearlyfull penetrance and occur spontaneously 50% of the time. NF1, with anincidence of 1 in 40,000 individuals is a much more rare disorder thenNF2, which has an incidence of 1 in 3,000.

[0023] Reflective of their very different clinical appearances, MF1 andNF2 are caused by two entirely different genes. The NF1 gene onchromosome 17 encodes a protein named neurofibromin. The function ofneurofibromin is unclear, although it does contain a single domain withGTPase activating activity. The NF2 gene on chromosome 22 encodes theMerlin protein. The exact function of Merlin is also unknown, but it ishighly homologous to a family of cytoskeleton associated proteinsincluding moesin, ezrin, radixin, and talin.

[0024] Amyotrophic lateral sclerosis (ALS) is the most commonlydiagnosed progressive motor neuron disease. The disease is characterizedby degeneration of motor neurons in the cortex, brainstem, and spinalcord. Generally, the onset is between the third and sixth decade. ALS isuniformly fatal, typically within five years. The cause of the diseaseis unknown although some cases of familial ALS map to the sod-1 gene.

[0025] Post mortem brains from ALS patients show affected neurons of thecerebral cortex, the anterior horns of the spinal cord, and thehomologues in some of the motor nuclei of the brain stem. The class ofneurons affected is highly specific: first motor neurons for ocularmotility, then later in the disease, sphincteric motor neurons of thespinal cord.

[0026] Although death occasionally results shortly after the onset ofthis symptomatic disease, the disease generally ends with respiratoryfailure secondary to profound generalized and diaphragmatic weakness.

[0027] Multiple Sclerosis (MS) is a neurodegenerative disease of thebrain and spinal cord in which a breakdown occurs in the myelinsheathing of the nerve fibers. MS is currently incurable and treatmentsare few and usually result in only temporary improvements of the diseasesymptoms.

[0028] Brain injury, stroke, and multiple infarct dementia are injuriesto the brain that have no necessary genetic component but often resultin similar neuronal deficits similar to those seen in the geneticdisorders described above.

[0029] Apolipoprotein E (ApoE) has been extensively studied innon-nervous tissues as one of several proteins that regulate lipidmetabolism and transport. ApoE facilitates cholesterol transport betweendifferent cell types and different organs. Specifically, it binds tolarge lipid-protein particles (called lipoproteins) and increases theirability to transport cholesteryl esters.

[0030] The mature form of ApoE, found in human plasma and cerebrospinalfluid (CSF), is a single, glycosylated, 37 kDa polypeptide containing299 amino acids. There is some evidence that ApoE may coordinate themobilization and redistribution of cholesterol in repair, growth, andmaintenance of myelin and neuronal membranes during development or aftersciatic nerve injury. In the brain, ApoE appears to coordinate theredistribution of cholesterol and phospholipids. Removal of the apoEgene by gene disruption or “knockout,” appears to cause someage-dependent reduction of synaptic contacts in the cortex andimpairment of reinnervation in the hippocampus.

[0031] In humans there are three major isoforms of ApoE (E4, E3 and E2)differing by a single unit of net charge which can be easily detected byisoelectrofocusing. These isoforms are expressed from multiple allelesat a single apoE genetic locus, giving rise to three common homozygousphenotypes (E4/4, E3/3 and E2/2) and three common heterozygousphenotypes (E4/3, E4/2, and E3/2).

[0032] The allele frequency of apoE4 is markedly increased in sporadicand late onset familial Alzheimer's disease (AD). As many as 80% ofclinical cases of AD (aged between 65 and 75 years) carry the E4 allelecompared to only 15% in the normal population.

[0033] Drugs for the treatment of patients with neurodegenerativedisease, such as Alzheimer's disease, are few in number despite the needfor such therapies. Only two FDA approved drugs are currently approvedfor treating symptoms of Alzheimer's disease: tacrine (Cognex™manufactured by Parke-Davis) and donepezil (Aricept™ marketed byPfizer).

SUMMARY OF THE INVENTION

[0034] In general, the invention features a method of preventing aneuronal deficit in a patient diagnosed with a neurological disease orpredisposition to a neurological disease. The method includesadministering to the patient a therapeutically-effective amount of acomposition capable of increasing ApoE levels. Preferably the compoundis one of the following: probucol, tacrine, heptylphysostigmine,simvastatin, lovastatin, pravastatin, thorvastatin, probucol analogs,vitamin E, donepezil, blood pressure inhibitors, antioxidants,anti-inflammatories, and steroids. Use of tacrine, donepezil, orestrogen alone for the treatment of Alzheimer's disease is specificallyexcluded. In one embodiment, AD and stroke are specifically excludedfrom the neurological diseases of the invention. Methods for thetreatment of Alzheimer's disease with tacrine, donepezil, or estrogenincludes administering another compound such as a second ApoE enhancingcompound or vitamin E. The therapeutically-effective amount of thecomposition used in the methods is a dosage which is sufficient toincrease ApoE levels in the patient being treated. Further, in oneembodiment the increase in ApoE levels is sufficient to also increaseamyloid scavenging.

[0035] In preferred embodiments, the patient has been identified ashaving at least one apoE4 allele; the patient has afflictions thataffect either the central nervous system (CNS), peripheral nervoussystem (PNS), or both; the patient has been diagnosed to suffer from, orhave a genetic predisposition to, a neurodegenerative disease such asAlzheimer's disease, Creutzfeldt-Jakob disease, Huntington's disease,Lewy body disease, Parkinson's disease, Pick's disease, amyotrophiclateral sclerosis, multiple sclerosis, neurofibromatosis, stroke,multiple infarct dementia, or brain injury.

[0036] In other embodiments, the invention provides a method fortreating presymptomatic as well as symptomatic patients and the methodmay include a preferred dosage administered orally but also may includeother drug delivery methods known to those skilled in the art.

[0037] The invention also provides a method for increasing ApoE levelsin a patient by administering a therapeutically-effective amount of acomposition selected from the group comprising probucol, tacrine,heptylphysostigmine, simvastatin, lovastatin, pravastatin, thorvastatin,probucol analogs, vitamin E, donepezil, blood pressure inhibitors,antioxidants, anti-inflammatories, and steroids. The increase in ApoElevels may be measured by quantitating ApoE polypeptide with an antibodyor by other methods known to those skilled in the art for quantitatingincreases in biological activity, gene expression, or protein levels. Invarious preferred embodiments, the patient has at least one apoE4allele; is suffering from or is genetically predisposed to suffer from adisease selected from the group consisting of Alzheimer's disease,Creutzfeldt-Jakob disease, Huntington's disease, Parkinson's disease,Pick's disease, Lewy body disease, amyotrophic lateral sclerosis,multiple sclerosis, and neurofibromatosis, or a disease without anecessary genetic component such as brain injury, stroke, and multipleinfarct dementia. The administration of the compounds for this purposemay be to enhance cognitive performance in both diseased and undiseasedindividuals.

[0038] In another aspect, the invention provides a method foridentifying a therapeutic compound effective for the treatment ofneurodegenerative disease by exposing a cell, for example an astrocyte,to a compound (or mixture of compounds) and measuring the expression ofthe apoE gene (e.g., by mRNA or polypeptide quantitation techniques) or,an increase in ApoE polypeptide levels. An increase in one of the aboveindices indicates a potential therapeutic compound. In a preferredembodiment, the method for identifying a therapeutic compound includesexposing a cell from the nervous system (for example, an astrocyte, anoligodendrocyte, a neuron, or a glial cell) to a compound and measuringthe expression of apoE. The cell may be adapted to cell culture or thecell may be derived from a patient afflicted with a neurodegenerativedisease (for example, Alzheimer's disease, Creutzfeldt-Jakob disease,Huntington's disease, Parkinson's disease, Pick's disease, Lewy bodydisease, amyotrophic lateral sclerosis, multiple sclerosis,neurofibromatosis, brain injury, stroke, or multiple infarct dementia).An increase in apoE or ApoE levels is indicative of atherapeutically-effective compound. Preferably, the increase is about10% or more.

[0039] For the purpose of the present invention the following terms aredefined below.

[0040] By “Alzheimer's Disease (AD)” is meant a pathology characterizedby an early and extensive loss of enterhinal cortex neurons. AD patientsmay be identified by progressive and degenerative effects on the brainwhich are not attributable to other causes. A diagnosis of Alzheimer'sdisease is made using clinical-neuropathological correlations known inthe art (see, e.g., Arch. Neurology, 51(9):888-896 (1994)). Post mortem,the disease may be diagnosed by the presence of amyloid plaques in thebrain.

[0041] By “non-AD neurological disease (non-AD)” is meant any diseaseother than Alzheimer's disease, which involves the neuronal cells of thenervous system. Specifically included are: prion diseases (e.g,Creutzfeldt-Jakob disease); pathologies of the developing brain (e.g.,congenital defects in amino acid metabolism, such asargininosuccinicaciduria, cystathioninuria, histidinemia,homocystinuria, hyperammonemia, phenylketonuria, and tyrosinemia, andfragile X syndrome); pathologies of the mature brain (e.g.,neurofibromatosis, Huntington's disease, depression, amyotrophic lateralsclerosis, multiple sclerosis, and stroke); conditions that strike inadulthood (e.g. Alzheimer's disease, Creutzfeldt-Jakob disease,Huntington's disease, Lewy body disease, Parkinson's disease, Pick'sdisease, amyotrophic lateral sclerosis, multiple sclerosis,neurofibromatosis, brain injury, stroke, and multiple infarct dementia);and pathologies of the brain (e.g., brain mishaps, brain injury, coma,infections by various agents, dietary deficiencies, and cardiovascularaccidents).

[0042] By “diagnosed to suffer from a neurological disease” is meantalready diagnosed as having the neurological disease, having a geneticpredisposition to the disease, or both.

[0043] By “genetically predisposed to suffer from a neurologicaldisease” is meant having a genetic predisposition to the disease, forexample, as determined by genotyping.

[0044] By “apoe allele load” is meant a determination of the type andnumber of apoE alleles present in the patient, whether determined bynucleic acid sequencing, examination of ApoE protein, or other methodsavailable to those skilled in the art.

[0045] By “neuronal deficit” is meant shortage or compromise of any ofthe conducting cells, support cells, or insulating cells of the centralor peripheral nervous system. This term may also refer to alteredcognitive aspects such as memory performance, verbal memory, spatialmemory, factual memory, or learning capacity. This term may also be usedto refer to compromised non-cognitive aspects of the nervous system, forexample, motor coordination, voluntary or involuntary neuronalresponses, muscle movements or reflexes.

[0046] By “treating” is meant the medical management of a patient withthe intent to cure, ameliorate, or prevent a disease, pathologicalcondition, or disorder. This term includes active treatment, that is,treatment directed specifically toward the improvement or associatedwith the cure of a disease, pathological condition, or disorder, andalso includes causal treatment, that is, treatment directed towardremoval of the cause of the associated disease, pathological condition,or disorder. In addition, this term includes palliative treatment, thatis, treatment designed for the relief of symptoms rather than the curingof the disease, pathological condition, or disorder; preventivetreatment, that is, treatment directed to prevention of the associateddisease, pathological condition, or disorder; and supportive treatment,that is, treatment employed to supplement another specific therapydirected toward the improvement of the associated disease, pathologicalcondition, or disorder. The term “treating” also includes symptomatictreatment, that is, treatment directed toward constitutional symptoms ofthe associated disease, pathological condition, or disorder.

[0047] By “therapeutically-effective amount” is meant an amount of apharmaceutical composition sufficient to produce a healing, curative, orameliorative effect either in the treatment of a neurological disease orin the prevention of a neurological disease.

[0048] By “probucol-containing composition” is meant any compositionthat includes, as a component, probucol.

[0049] By “tacrine-containing composition” is meant any composition thatincludes, as a component, tacrine.

[0050] By “therapeutically-effective” is meant that a compound shallincrease ApoE protein, as measured as secreted protein circulating inthe cerebrospinal fluid, by a measurable amount, as compared to age anddisease matched control patients.

[0051] By “amyloid scavenging” is meant that a therapeutically effectivecompound shall increase the amount of amyloid degradation, uptake, orclearance such that the amount of amyloid material is reduced.

[0052] The present invention provides two important advantages. First,it provides a therapy for prophylactically halting or reducingneurodegeneration in a patient predisposed to acquire aneurodegenerative condition and, second, the invention can reverse,halt, or slow the progression of a diagnosed disease. Further, theprobucol-containing compounds utilized herein are non-toxic,pharmocokinetically well understood, and are known to be well toleratedby mammals. Finally, probucol has the advantage of directly saving andrestoring the health of a neural tissue rather than being a merepalliative.

DETAILED DESCRIPTION OF THE INVENTION

[0053] The drawings will first be described.

BRIEF DESCRIPTION OF THE DRAWINGS

[0054]FIG. 1 is a schematic representation of the postulated cascade ofevents regulating cholesterol transport during CNS reinnervation (FC:free cholesterol; CE: cholesterol esters; and E: ApoE).

[0055]FIG. 2 is a graph depicting the difference in latency performancein a Morris swimmaze for a 3 month old control mouse versus aheterozygote apoE knockout mouse.

[0056]FIG. 3 is a histogram depicting the cholinergic status in thebrains of control (solid bars) and Alzheimer's disease subjects (hashedbars) with different apoe genotypes (*:p<0.05;***:p<0.001).

[0057]FIG. 4 is a graph depicting brain ApoE levels in Alzheimer'sDisease (AD) subjects as a function of the apoE genotype (*:p<0.05versus apoE3/3 AD subjects). Mean age-matched control value isrepresented by the solid bar (±SEM).

[0058]FIG. 5 is a histogram depicting the effect of variousconcentrations of tacrine on secreted ApoE levels in rat type-1astrocytes in culture.

[0059]FIG. 6 is a drawing showing the molecular structure of probucol.

[0060]FIG. 7 is a immunoblot showing the effect of different doses ofprobucol on ApoE secretion and steady state levels in the media of invitro cultured rat type 1 astrocytes.

[0061]FIG. 8 is a graph that shows the dose response of ApoE levels indifferent murine tissues following 10 days of treatment with probucol.

[0062]FIG. 9 is a graph representing the change in the ADAS-Cog Scores(70 pt. Scale) at six months as a function of change in ApoE levels inthe CSF measured at one month.

[0063]FIG. 10 is a graph representing a linear regression analysis thatshows the change in ADAS-Cog scores (117 pt. extended scale) after threemonths as a function of change in ApoE protein levels in the CSF after 1month (p>0.05; r²=0.20).

[0064]FIG. 11 is a graph representing a linear regression analysis thatshows the change in ADAS-Cog scores (117 pt. extended scale)after sixmonths as a function of change in ApoE protein levels in the CSF at onemonth (p>0.05, r²=0.19; excluding patient PP 105 p<0.02, r²=0.72).

[0065]FIG. 12 is a graph representing a linear regression analysis thatdepicts the relationship between the change in lipid peroxidation (%baseline) as a function of a change in ApoE levels.

[0066]FIG. 13 is a graph representing a linear regression analysis thatdepicts the relationship between the change in ADAS-Cog scores (117 pt.extended scale; six months-baseline) as a function of change in lipidperoxidation (% baseline).

[0067]FIG. 14 is a graph representing a linear regression analysis thatdepicts the relationship between the change in ADAS-Cog scores (70 pt.scale; six months—baseline) as a function of change in lipidperoxidation (% baseline).

[0068] The invention described herein features a method for promotingneuroregeneration and repair.

[0069] The invention is based on our discovery that someneurodegenerative diseases are related, in part, to abnormal ApoEbiology.

[0070] Certain apoE alleles, such as the apoE4 allele, encode unstableApoE gene products that compromise nerve biology. This eventuallyresults in a nervous system deficit or exacerbates a nervous systemdisease as evidenced by impaired cognitive, sensory, or motor function.We have made an important connection between the molecular pathwaywithin which ApoE works, nerve repair, and how drugs that affect ApoElevels will improve neurological disease symptoms. We propose that aselective dysfunction of the lipid transport system controlled by ApoEcould be central to the pathophysiological process that characterizesApoE4 carrying AD subjects. In addition, we have discovered thatincreasing ApoE levels improves amyloid scavenging and this may explainobserved improvements in animals treated with compounds that can raiseApoE levels. We have conceived of a way to increase ApoE levels usingexisting drugs, such as probucol and tacrine. We propose thatupregulating ApoE levels in AD patients may improve neuro-regenerationand this is supported by our observations that both probucol and tacrineincrease ApoE levels. Thus, our invention provides a way to directlyrescue dying cells of the nervous system by replenishing unstable ApoEprotein or enhancing ApoE levels by inducing apoE gene expression byadministration of these drugs. The invention further provides for theuse of probucol, tacrine, and other ApoE elevating compounds to treat ADand non-AD neurodegenerative disease.

[0071] Therefore, as described herein, probucol has importanttherapeutic properties. Probucol is predicted to improve brain ApoEexpression for all patients and to have especially therapeutic effectsin patients with genotypes predictive of very low or unstable ApoEexpression (e.g., patients having apoE 4/3 and 4/4 allele loads). Wepredict that other drugs such as heptylphysostigmine, simvastatin,lovastatin, pravastatin, thorvastatin, probucol analogs, vitamin E,donepezil, blood pressure inhibitors, antioxidants, anti-inflammatories,and steroids, will alter ApoE levels and have similar therapeutic healthbenefits. We also provide methods for readily determining thetherapeutic effectiveness of such compounds for use in the methods ofthe invention.

[0072] In addition, the results described herein demonstrate thatsymptoms of neurodegenerative disease may be exacerbated by low ApoElevels in the brain and that these symptoms will be reduced by increasesin ApoE levels. Symptoms of AD and non-AD patients correlate withalterations in ApoE levels and are improved with probucol. Theimprovement is similar to that observed in AD patients treated withtacrine. The present invention therefore provides methods and reagentsfor the treatment of AD and non-AD diseases, such as Creutzfeldt-Jakobdisease, Huntington's disease, Lewy body disease, Parkinson's disease,Pick's disease, amyotrophic lateral sclerosis, neurofibromatosis, braininjury, stroke, multiple sclerosis, and multiple infarct dementia.

[0073] Below we provide experimental data showing the ability of thedrugs provided above to increase ApoE expression in cells of the nervoussystem.

[0074] Further, our invention provides a clinical trial to demonstratethat patients with a neurodegenerative disease can be effectivelytreated with these drugs.

[0075] Introduction

[0076] Eukaryotic cells obtain their cholesterol from two distinctsources: a) it is synthesized directly from acetyl-CoA through theHMG-CoA reductase pathway or, b) it is imported through the apoE/apoB(LDL) receptor family via lipoprotein-complex internalization. These twodifferent pathways are tightly coupled: i.e., a reduced internalizationof cholesterol through the receptor pathway will cause increases inHMG-CoA reductase activity (cholesterol synthesis) whereas inhibition ofintracellular cholesterol synthesis will induce expression of the LDLreceptor and lipoprotein internalization.

[0077] Much of the free cholesterol generated during synapse degradationis stored in astrocytes in the central nervous system (CNS) and, inmacrophages in the peripheral nervous system (PNS) where it iseventually reused during PNS regeneration and CNS reinnervation.Following binding of the ApoE complexes with neuronal LDL receptors, theApoE/cholesterol/LDL receptor complex is internalized, degraded and thecholesterol released inside neurons is used for membrane synthesis aswell as synaptic remodeling (FIGS. 1, 3, and 4). The increase inintracellular neuronal cholesterol levels causes a down-regulation ofHMG-CoA reductase activity and mRNA prevalence in granule cell neuronsundergoing dendritic and synaptic remodeling (FIGS. 1 and 7). Followingneuronal cell loss and terminal deafferentation in the brain, largeamounts of lipids are released from degenerating axon membranes andmyelin (FIG. 1, Step 1). In response, astrocytes (FIG. 1, Step 2) andmacrophages synthesize and release ApoE within the lesion to scavengecholesterol from both cellular and myelin debris. Using the activity ofthe 3,3-hydroxy-methylglutaryl-CoA reductase (HMG-CoA reductase) tomonitor cholesterol synthesis, it was found that a critical phase ofcholesterol synthesis is progressively repressed in response to theincrease in intracellular cholesterol concentration. Thus, cholesterolrequired for membrane synthesis can reach critically low levels duringperiods of neuronal cell injury. Our discovery shows that increases inApoE synthesis can address this shortage.

[0078] The ApoE Link to Non-AD Diseases

[0079] We believe that AD patients who are apoE4 carriers arecharacterized by a defective system of repair due to lower ApoE brainlevels. Accordingly, drugs like tacrine and more importantly, probucol,could be used to increase ApoE levels in the brain of apoE4 carriers,improve lipid transport and delivery, and stimulate reinnervation andsynapse replacement generally. We believe that it is possible to eitherstop or slow down the disease process in both AD and non-AD patients byincreasing the amount of functioning ApoE.

[0080] Although, the role of ApoE4 has been best studied in Alzheimer'sdisease, we believe ApoE is crucial for the functional recovery andenhanced survival of neurons in non-AD neurodegenerative diseases.Hence, regulating ApoE levels may alter the rate of progression orinfluence the age of onset and/or death of a host of neurodegenerativediseases. Thus, pharmaceuticals that allow for the manipulation of ApoElevels will be useful reagents for the treatment of manyneurodegenerative diseases.

[0081] We predict that drug responsiveness may be dependent upon theApoE phenotype in Alzheimer's disease. Because of the role of ApoE inreinnervation and synaptic replacement in the brain, we postulate thatthe presence of the ApoE4 will not only affect drug responsiveness inAlzheimer's disease, but that it will also affect drug responsiveness inother neurodegenerative diseases of the CNS, particularly where apoE4allele load has been shown to affect symptomology.

[0082] We believe that the observed reduction in brain ApoE levelsreported in apoE4 carriers results in defective delivery of cholesteroland phospholipids into neurons. In this model, the LDL receptor familyis playing a crucial role in mediating the internalization of the ApoElipoprotein-complexes. The internalized lipids indeed constitute thebuilding blocks for the formation of membrane, terminal andsynapto-dendritic structures. We hypothesize that the impaired deliveryof major membrane lipids in E4 carriers compromises synaptic integrity(as reported by Masliah et al., 1995), pre-synaptic terminal strictures,(as indicated by the losses of ChAT activity in the hippocampus andcortex of apoE4/AD subjects) ultimately leading to impaired cognitiveperformance.

[0083] Drugs Suitable for Increasing ApoE Levels

[0084] The methods provided herein make use of probucol (Lorelco™)prepared by and commercially available from Marion Merrell Dow Inc.Nonetheless, we predict that the following other drugs namely, tacrine(Cognex™, manufactured by Parke-Davis), heptylphysostigmine(manufactured by Merck Corp.), simvastatin (Zocor™, manufactured byMerck Corp.), lovastatin (Mevacor™, manufactured by Merck Corp.),pravastatin (Pravachol, manufactured by Bristol-Myers Squibb),thorvastatin (Parke-Davis), probucol analogs, vitamin E, donepezil(Aricept™), blood pressure inhibitors, antioxidants,anti-inflammatories, and steroids are suitable for altering ApoE levelsand eliciting similar beneficial effects for the treatment of theafflictions described herein. The molecular structure of probucol isprovided in FIG. 6. The compounds described above are all commerciallyavailable and have established dosage protocols.

[0085] It will be appreciated that other compositions which raise ApoElevels may be identified using the assays provided herein.

[0086] We predict that the primary efficacy of these compounds andcompositions rests with their ability to increase levels of ApoE. Wehave developed an efficient bioassay to screen for useful drugs capableof increasing ApoE levels. A cell culture system using an primary ratastrocyte cell line is exposed to varying concentrations of probucol orany other potential drug and ApoE levels are measured at the mRNA levelor protein level. Other culture systems which may be used include humanculture systems of primary cells derived from human tissue orimmortalized cell lines of nerve tissue origin such as the humanastrocyte cell line, AsCH-7 (Bobryshev et al., 1995). In addition, novelcell lines from patients with various apoE genotypes may be used, aswell as cell lines from patients suffering or predisposed to suffer fromany of the neurodegenerative diseases described herein. Adult spinalcord from post-mortem patients with any of the above diseases may beused as a source for deriving cell lines of nerve tissue origin. Onemethod of immortalizing cells from these patients is to infect primarytissue with a replication-deficient retrovirus containing DNA sequencesencoding the SV40 large T antigen (Whittemore et al., 1994). Anothermethod is to derive immortal human-human hybrid cell lines that expressphenotypic characteristics of primary oligodendrocytes or astrocytes byfuising a 6-thioguanine-resistant mutant of the human rhabdomyosarcomaRD cell line with adult human nerve tissue by a lectin-enhancedpolyethylene glycol procedure (McLaurin et al., 1995). Other methods forimmortalizing cells derived from primary human nerve tissue couldinclude the use of chemical carcinogens or radiation. The use of animalmodels such as mice with mutations or gene disruptions of any genesknown to contribute to any of the diseases described herein is also asource of nerve tissue to be used in a cell based assay for thescreening of drugs capable of increasing ApoE levels. Immortalized linesobtained in this manner would possess the genetic lesion of the diseasein question and provide a source for screening ApoE inducingpharmaceuticals uniquely suited for that particular neurodegenerativedisease. Antibodies to ApoE allow for the design of a high throughputassay using an immunoassay detection method.

[0087] One particular application of this assay would be to evaluate thepotency of probucol analogs for inducing ApoE expression. These analogsof probucol lack the anti-oxidant properties of probucol and wouldeither lack the hydroxyl group/s (OH) or contain a modified hydroxylgroup/s. In general, methods for producing probucol analogs include thecondensation of 4-mercapto-2,6-disubstituted phenols with acetone. Thesynthetic yields for probucol are approximately 90% while the yields forother analogs are variable but normally greater than 40%. Such analogsprovide an opportunity to dissociate the anti-oxidant properties ofprobucol from the ApoE inducing properties of probucol. If probucolanalogs increase ApoE levels in the cellular screening assay, then thiswould demonstrate that ApoE induction is an antioxidant independentmechanism.

[0088] Further, we have observed that compounds such asheptylphysostigmine, tacrine, and various HMG Co-A reductase inhibitorsincrease the levels of extracellular ApoE in the astrocyte screeningassay. In contrast, the specific acetylcholinesterase inhibitordonepezil had no effect on secreted ApoE levels. These results indicatethat a variety of known drugs can modulate the extracellular levels ofApoE in an antioxidant independent way and suggest that the antioxidantproperties of probucol may not be critical to the mechanism responsiblefor increases in ApoE.

[0089] Administration of Probucol-Containing and Tacrine-ContainingCompositions

[0090] Probucol-containing and tacrine-containing compounds, such asLorelco™ and Cognex™ are commercially available from Marion Merrell DowCo. and Parke-Davis, respectively. The. clinical dosage form for oraladministration of Lorelco™ is either a 250 mg or 500 mg tablet.Preferably, tablets are administered at a dosage of at least 500 mgtwice daily by oral administration. Cognex is available in the form of a10 mg or 40 mg capsule and can be titrated up to 160 mg per day by oraladministration.

[0091] Other formulations for treatment or prevention of the conditionsdescribed herein, may take the form of a compound that alters ApoElevels and may be combined with a pharmaceutically-acceptable diluent,carrier, stabilizer, or excipient. Conventional pharmaceutical practiceis employed to provide suitable formulations or compositions toadminister such compositions to patients. Oral administration ispreferred, but any other appropriate route of administration may beemployed, for example, parenteral, intravenous, subcutaneous,intramuscular, intracranial, intraorbital, ophthalmic, intraventricular,intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal,or aerosol administration. Therapeutic formulations may be in the formof liquid solutions or suspensions (as, for example, for intravenousadministration); for oral administration, formulations may be in theform of liquids, tablets or capsules; and for intranasal formulations,in the form of powders, nasal drops, or aerosols.

[0092] Methods well known in the art for making formulations aredescribed, for example, in “Remington's Pharmaceutical Sciences,” MackPublishing Company, Easton, Pa. Formulations for parenteraladministration may, for example, contain excipients, sterile water, orsaline, polyalkylene glycols such as polyethylene glycol, oils ofvegetable origin, or hydrogenated napthalenes.

[0093] If desired, slow release or extended release delivery systems maybe utilized. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds. Otherpotentially useful parenteral delivery systems include ethylene-vinylacetate copolymer particles, osmotic pumps, implantable infusionsystems, and liposomes. Formulations for inhalation may containexcipients, for example, lactose, or may be aqueous solutionscontaining, for example, polyoxyethylene-9-lauryl ether, glycocholateand deoxycholate, or may be oily solutions for administration in theform of nasal drops, or as a gel.

[0094] In general, probucol or tacrine, or other compounds for use inthe methods of the invention are administered at a dosage appropriate tothe effect to be achieved and are typically administered in unit dosageform. As noted above, the preferred route of administration for mostindications is oral.

[0095] An effective quantity of a probucol-containing,tacrine-containing, or the other compound of the invention is employedto treat the conditions described herein. The exact dosage of thecompound may be dependent, for example, upon the age and weight of therecipient, the route of administration, and the severity and nature ofthe symptoms to be treated. In general, the dosage selected should besufficient to prevent, ameliorate, or treat the condition, or one ormore symptoms thereof, without producing significant toxic orundesirable side effects.

[0096] The bioassays described in Example 9 can also be employed indetermining dosage for known and unknown drugs such that atherapeutically effective amount necessary to increase ApoE levels incells of nerve tissue origin could be determined.

[0097] In the case of probucol, there is no acute toxicity in mice orrats. A single case of an overdose in a human was resolved by inducedvomiting and the patient had no lasting ill effects.

[0098] The following detailed examples are provided for the purpose ofillustrating, and not limiting, the invention.

EXAMPLE 1 ApoE-/—Mice Exhibit Neuronal Deficits

[0099] Homozygous apoE-deficient (knockout) mice display a significantloss of synapses and marked disruption of the dendritic cytoskeletonwith normal aging. More importantly, these animals failed to showcompensatory synaptogenesis following lesions of entorhinal cortexprojections. The importance of ApoE in brain lipid transport is furtherunderscored by the absence of other key plasma apolipoproteins such asapoA1 and apoB in brain tissues.

[0100] Analysis of spatial memory performance in the Morris swimmaze often apoE knockout mice and ten control mice cross-bred to homogeneity ina C57B1 genetic background revealed marked impairment of cognitiveperformance in 3, 5, and 6 month old animals. These geneticallystabilized apoE knockout mice (˜99% pure C57B1 background) are derivedfrom the North Carolina apoE knockout mice colony. Briefly, mice(control and heterozygote apoE knockouts) were placed into a 1.6 mdiameter pool filled (45 cm depth) with water made opaque by theaddition of powdered skim milk. The animals must locate a platformsubmerged 2 cm below the surface of the water. Animals finding theplatform within 120 sec were allowed to stay on the platform for 30 secand then removed. Each mouse was given 4 trials per day for four days oftesting. The primary dependent variables measured are the latency anddistance to find the platform. FIG. 2 summarizes the latency resultsobtained in young (3 months old) homozygote apoE knockout mice versuscontrol mice. Detailed analyses of swimming speed and pattern as well asvisual deficits revealed no impairment in either motor or visualcoordination in the knockout mice.

[0101] These results indicate synaptic integrity and plasticity, as wellas spatial memory performances, are seriously compromised by the absenceof ApoE in the brain. Interestingly, regeneration and plasticity arerelatively intact in the injured PNS in apoE knockout mice where, incontrast to the CNS, apoB, apoJ and apoA1 behave like back-up systemsfor the transport of lipids during neural tissue remodeling.

EXAMPLE 2 Apolipoprotein E4 and Alzheimer's Disease

[0102] Alzheimer's disease pathology is characterized by an early andextensive loss of entorhinal cortex neurons and a marked loss of neuronsin the hippocampal CA1 area. However, in contrast to the entorhinalcortex lesioned rat brain, only a small number of AD patients (withundetermined apoE genotype) are capable of displaying synapticplasticity in response to hippocampal deafferentation. It has also beenshown that there are significant decreases in presynaptic terminaldensity (−45%) in the neocortex and in layer V of the frontal (−27%) andtemporal cortices (−36%). We predict that these pathologies observed inAD are linked to abnormal ApoE metabolism.

[0103] Indeed, the link between ApoE metabolism and AD is supported bygenetic findings that demonstrate overlap with ApoE neurobiology and AD.The frequency of the apoE4 allele was shown to be markedly increased insporadic as well as in late onset familial Alzheimer's disease. A genedosage effect was observed in both familial and sporadic cases: i.e., asage of onset increases, apo E4 allele copy number decreases.

[0104] The apoE4 allele prevalence analyses in both living (probable) ADpatients and autopsied (confirmed) AD subjects from Eastern Canadaindicate that as many as 80% of all our AD subjects, aged 65 to 75, arecarriers of at least one copy of the apoE4 allele. The apoE4 alleledosage also is observed to modulate the age of onset in AD families withthe amyloid precursor protein (APP) mutation on chromosome 21 and in ADfamilies with mutations in the presenilin 2 gene on chromosome 1.Families with chromosome 14-linked AD are apparently not affected by theapoE4 allele.

[0105] The apoE4 allele copy number was also shown to have an impacton: 1) the risk of developing AD; 2) the accumulation of mature senile(amyloid) plaques and tangles in the cortex and hippocampus; 3)cholinergic cell density in the basal forebrain; 4)cholineacetyltransferase activity and nicotinic receptor binding sites in thehippocampus and temporal cortex, and; 5) neurofibrillary tanglesdensity.

[0106] These results indicate the existence of a very close relationshipbetween the apoE4 allele dosage and the neuropathology of AD. Forexample, AD subjects carrying at least one copy of the apoE4 alleleexhibit a marked reduction of synapse density when compared to apoE3/3AD subjects or to age-matched control individuals.

EXAMPLE 3 ApoE4 and Cholinergic Dysfunction in AD

[0107] Brain membrane phospholipids, especially phosphatidylcholine (PC)and phosphatidylethanolamine (PE), have been shown to be involved in theavailability of choline, a rate-limiting precursor of acetylcholine(ACh). The release from PC of free choline precursor for ACh synthesisis accomplished in a one step process through a phospholipase-D typeenzyme in cholinergic neurons. Brain levels of choline are decreased byup to 40-50% in frontal and parietal cortices of AD patients whereascholesterol, which is required for the proper functioning of thenicotinic receptor sub-type, is markedly reduced in AD versus controlsubjects.

[0108] As losses of cholinergic neurons and/or choline acetyltransferase(ChAT) activity are well known neurochemical hallmarks of AD, therelationship between the apoE4 genotype and cholinergic deficits ishighly relevant to investigate in genetically distinct individuals.Thus, we quantified cholinergic neuron density in the nucleus basalis ofMeynert and in the diagonal band of Broca in post-mortem AD subjectswith and without the apoE4 allele. We also examined the impact ofcholinergic cell reduction on cholinergic markers in the brain areasthat receive cholinergic input from these brain areas, namely, thehippocampus and the cortex.

[0109] The cholinergic markers examined were the cholineacetyltransferase activity (ChAT: essentially pre-synaptic) andnicotinic receptor (NR: largely pre-synaptic) in control and AD subjectscarrying 0 (E3/3), 1 (E4/3) or 2 (E4/4) copies of the apoE4 allele. FIG.3 summarizes our findings. Although reductions in cholinergic neurondensity were found in apoE4 and non-apoE4 AD subjects at the level ofthe nucleus basalis of Meynert (middle graphs) and the diagonal band ofBroca, only AD subjects carrying 1 or 2 copies of E4 showed significantloss of pre-synaptic ChAT and NR in the hippocampus and temporal cortex.In other words, apoE3/3 AD subjects, despite a 60 to 70% cell loss inthe basalis, manage to maintain cortical and hippocampal ChAT activitiesand NR densities within normal range.

[0110] These results are consistent with the theory that the apoEgenotype may directly influence the synaptic plasticity of thecholinergic system in response to neuronal cell loss. In this model,ApoE4 compromises lipid homeostasis and consequently impairs membraneremodeling. These results demonstrate that cholinergic reinnervation andsynaptic plasticity are markedly compromised in several brain areas inAlzheimer's disease subjects carrying 1 or 2 copies of apoE4.Furthermore, when the extent of the regeneration potential of E4 andnon-E4 carriers was quantified, a massive reduction in the overallregenerative capacity of apoE4 subjects in at least six different brainregions was observed.

EXAMPLE 4 apoE4 Carriers and ApoE Concentrations in the Brain

[0111] We have observed that there is a relationship between synapticintegrity, reinnervation in the brain, and ApoE levels and this issupported by two otherwise disparate observations: a) the completeabsence of ApoE compromises synaptic density, reinnervation andplasticity and, b) successful synaptic remodeling in the intact animalis associated with the over expression of ApoE in the deafferented zone.

[0112] In the AD brain, apoE mRNA was shown to be present but notincrease 10 in response to cell loss and deafferentation. FIG. 4illustrates levels of ApoE measured in the brain of AD subjects withdifferent genotypes as well as in control subjects with apoE3/2 andapoE3/3 genotypes. Of the ˜90 neuropathological elderly control subjectsthat we have examined so far (using standard criteria), none of thosefitting the “CONTROL” criteria were found to carry the E4 allele. Asignificant reduction in ApoE concentration in tissue was measured inapoE4 carriers versus non-E4 subjects.

[0113] We have discovered that the risk of developing AD, theaccumulation of senile plaques and tangles in the AD brain, and the lossof cholinergic function in AD follow the exact same genotype gradient asthe concentration of ApoE measured in the brain of AD subjects: i.e.,increasing marked deterioration is observed in genotypes E2/2, E3/3,and, E4/4, the latter genotype representing the worse case scenario. Webelieve that the most important observation among these is the fact thatthe Alzheimer pathology is much more severe (cell loss, deafferentation,and increased GFAP expression) in apoE4 carriers having AD than it is inthe non-apoE4 AD subjects. Yet, ApoE levels, which should be increasedin response to damage and cell loss, are in fact decreased in those sameApoE4 individuals. We believe this decrease points to an underlyingmechanism of the pathology of neurodegenerative disease.

EXAMPLE 5 Pghophysiological Consequences of Low ApoE Levels

[0114] To address the pathophysiological consequences of having low ApoElevels in life long apoE4/AD carriers we examined apoE/apoB (LDL)receptor expression (which correlates with cholesterol internalization)and HMG-CoA reductase expression (cholesterol synthesis) in thehippocampus AD and control subjects with different apoE genotypes. Thiscomparison allows us to assess the consequences of the poor delivery oflipids in apoE4 carriers.

[0115] Table 1 summarizes the mRNA prevalence measured for the LDLreceptor, HMG-CoA reductase and glial fibrillary acidic protein (GFAP)in post-mortem brains of control and AD subjects with known apoEgenotypes. Beta-tubulin was used as a control transcript to adjust forRNA loading in the gels. GFAP mRNA prevalence, a well known marker oflocal tissue damage is markedly increased in apoE4 carriers whencompared to apoE3/3 AD subjects. This supports the theory that moresevere damage occurs in the brain of apoE4 carriers. The LDL receptorand HMG-CoA reductase mRNA prevalence were found to be increased in ADsubjects carrying the apoE4 allele when compared to non-E4 carriers.TABLE 1 mRNA Levels of GFAP, LDL, and HMG-CoA in Post Mortem BrainSamples of Patients with AD mRNA Prevalence in the Hippocampus of ADPatients ApoE3/3 ApoE4/3 ApoE4/4 Genes % OF CONTROLS GFAP 110 365* 345*LDL 86.5 173* 170* HMG-CoA 87 191* 152  N = 6 N = 7 N = 3

[0116] The reduction of total brain ApoE protein in apoE4 carriers issufficient to shift cholesterol metabolism from its normal steady stateconcentration to a depleted state. This forces cells to attempt tocompensate by upregulating both synthesis and internalization. Thus webelieve that ApoE depletion observed in the brain of apoE4 carriers maysignificantly compromise lipid delivery in the CNS in a manner whichcannot be compensated for by the usual compensatory mechanisms.Ultimately, the damage to nerve tissue is thus exacerbated. Theseresults provide us with an entirely novel explanation for the reportedreduction of cholesterol in the brain of AD subjects with unknowngenotype.

EXAMPLE 6 Alteration of Apo E Levels as the Basis of a New TherapeuticalApproach for AD

[0117] It has been recently demonstrated that chronic (1 month)intraventricular infusion of purified human ApoE3 and ApoE4 in apoEknockout mice effectively restored microtubulin-associated protein 2 andsynaptophysin-like immunoreactivities in the cortex of homozygoteknockout animals. This result indicates that both ApoE4 and ApoE3 areeffective in restoring dendritic integrity in the ApoE knock-out mice.More importantly, the effect of intracerebral infusion of ApoE3 andApoE4 on cognitive performance and spatial memory function in the apoEknockout mice was examined and it was observed to be improved. This isconsistent with the fact that ApoE4 is as good as ApoE3 at binding tothe LDL receptor and carrying lipids in the serum. However, in contrastto ApoE3, ApoE4 concentration is much lower in the serum and in thebrain of apoE4 carriers.

[0118] From these observations we concluded that manipulation of ApoEconcentrations in the brain of mammals will effectively restore synapticplasticity and reinnervation in the brain. The results summarized aboveindicate that there is nothing particularly toxic or detrimental withthe ApoE4 isoform, but rather that the metabolism of ApoE4 leads tolower concentration of ApoE in the brain of E4 carriers, as compared tonon-E4 carriers.

EXAMPLE 7 Proof of Principle of this Therapeutical Approach forAlzheimer's Disease Using Tacrine

[0119] Using astrocyte cell cultures, we recently screened severalAlzheimer- and cardiovascular-related drugs which are known to affectlipid metabolism in vivo for their effect on ApoE levels. One knownantidementia drug, called tacrine or “Cognex™” (manufactured byParke-Davis), was found to act as an inducer of ApoE synthesis and ApoEsecretion in astrocytes in culture. Tacrine is one of two FDA approveddrugs to treat Alzheimer's disease in the U.S. It improves memory andlearning functions in Alzheimer's disease by inhibiting acetylcholinedegradation (acetylcholinesterase inhibitor). We conclude from ourresults that tacrine achieves its beneficial effect by this effect onApoE synthesis.

[0120] Importantly, tacrine has been shown to delay institutionalizationby more than 1.5 years in Alzheimer's patients treated for a very longperiods of time. These results are interpreted as evidence for abeneficial effect of tacrine on the rate of deterioration of Alzheimer'sdisease that occurs more or less independently from cholinergic effecton memory performance. We propose that the effects of tacrine onlipoprotein homeostasis indicates that tacrine may affect lipidhomeostasis in the brain by interacting with esterases that control HDLhomeostasis and delivery to brain cells. This mode of action of tacrinein the CNS may contribute to the observed long-term beneficial effect oftacrine on slowing down disease progression in AD.

[0121] To verify this working hypothesis, we have examined the effect ofvarious concentration of tacrine on ApoE synthesis and secretion inbrain cells in culture. FIG. 5 illustrates the effect of tacrine on ApoEsecretion in rat type 1 astrocyte cultures exposed (for 8 hours) tophysiological and pharmacological concentrations of the drug. Tacrineincreases ApoE secretion in astrocytes at concentrations typically foundin the CSF of AD patients treated for several weeks with tacrine (2 uM).We recently examined cerebrospinal fluid concentration of ApoE in livingAD subjects treated with tacrine for several months and found higherlevels of ApoE (N=4: nearly significant despite small sample size) inthe drug treated patients compared to untreated AD subjects (Table 2).Experimental methods used to monitor ApoE levels are described inBertrand et al., Mol. Brain Res. 33:174-178 (1995). TABLE 2 ApoE Levelsin the CSF of Patients with AD Treated and Untreated with Tacrine ApoEConcentration (mg/dL) In the Cerebrospinal Fluid (CSF) ControlsAlzheimer + p values (Age Matched) Alzheimer Tacrine A vs A + T Mean0.42 0.36* 0.41 *0.09 S.E.M. 0.02 0.02 0.06 N = 16 30 4

[0122] In conclusion, while it is known that tacrine improves memory inAD patients and delays institutionalization by 1.5 years we have madethe novel discovery that the mechanism by which this drug works is tocause ApoE synthesis and secretion in vitro at physiologicalconcentrations in brain cells and finally, to cause a raise incerebrospinal fluid concentration of ApoE in living patients and to slowdown disease progression to a point where it delaysinstitutionalization. These observations suggest that the effect oftacrine on Alzheimer's disease progression is caused by its action onApoE synthesis and secretion. This observation is entirely consistentwith the results obtained in the memory impaired apoE knockout miceexposed to physiological or intracerebral levels of purified ApoE.

[0123] On that basis we claim that agents with properties like tacrine,such as probucol, heptylphysostigmine, simvastatin, lovastatin,pravastatin, thorvastatin, probucol analogs, vitamin E, Aricept, bloodpressure inhibitors, antioxidants, anti-inflammatories, and steroids maybe capable of inducing ApoE synthesis and secretion in brain cells, andtherefore represent very potent promoters of synaptic plasticity andreinnervation in the normal brain. We forecast they should slow downAlzheimer's disease progression and delay the arrival of the Alzheimer'sdisease in those who are at risk but not yet symptomatic. These agentsshould also have therapeutic effects in other neurodegenerative diseasewhere synaptic plasticity and reinnervation are at least in partcompromised by the disease state.

EXAMPLE 8 Probucol for the Treatment of Neurodegenerative Disease

[0124] Several drugs were developed over the past decade to alter bloodlipid content in individuals who are at risk of developingcardiovascular diseases. One of these drugs, probucol (see FIG. 6), wasdeveloped in the 1960s by Dow Chemical Co. as a cholesterol loweringagent. Probucol is a very safe compound that also exhibits potentantioxidant properties. It was shown recently that subjects carrying theapoE4 allele were more responsive to the cholesterol lowering activityof probucol than subjects not carrying this allele. Circulating ApoElevels were shown to be increased in probucol-treatedhypercholesterolemic patients. Probucol was shown to be totallyuneffective in subjects with a very high physiological concentration ofApoE that exhibit the apoE2/2 genotype. Probucol is also known to lowertotal plasma apoB, apoA-1, apoA-II, apoC-II and apoC-III, all of whichhave little or no role to play in brain lipid homeostasis. The effect ofperipheral administration of probucol on CNS apolipoproteins is unknownin human.

[0125]FIG. 7 illustrates results obtained from rat primary type 1astrocyte cultures exposed to probucol at concentrations of 1 nM, 100nM, and 10 uM for 24 and 48 hours. Apo E concentration was assessed inthe cell culture medium by immunoblot analysis using methods previouslydescribed (Poirier et al., Neuroscience, 55:81-90 (1993)). In theabsence of probucol (EtOH control) very little ApoE is detected in themedia (i.e., the extracellular compartment). In contrast, concentrationsof probucol as low as 1 nM dramatically increased the extracellularamount of ApoE at 24 hours post dosing. Further increases in ApoE wereobserved with increasing concentrations of probucol. We also observedthat the amount of ApoE detected in the media at 48 hours was less thanthe amount of ApoE in the media after only 24 hours of incubation. Thistime-dependent response in ApoE levels may reflect the continueddivision and growth of cells.

[0126] In summary, in a manner similar to tacrine, pharmacologicalconcentrations of probucol were shown to cause marked synthesis andsecretion of ApoE in the astrocyte cultures media. These in vitroresults indicate that astrocytes, the cells responsible for thesynthesis of ApoE in the brain, secrete large amounts of ApoE whenexposed to probucol in vitro.

EXAMPLE 9 In Vivo Model for Determining the Effect of Probucol on ApoElevels

[0127] To extend the foregoing in vitro studies, the following animalstudy was conducted. Mice were dosed (i.p.) with probucol daily for tendays at doses ranging from 0.1 to 5 mg. At the end of ten days, theanimals were sacrificed and the brain tissue collected, rapidlydissected, and the hippocampi were isolated. The livers, tissue, andblood were also collected. Tissue homogenates from all ten animals werepooled and the ApoE levels in the different tissues were determined byimmunoblot. The intensity of the bands on the immunoblot werequantitated by scanning densitometry and these results are summarized inTable 3 and graphically represented in FIG. 8.

[0128] In animals treated with probucol (highest dose), ApoE levelsincreased dramatically in brain tissue but not in other tissues tested(i.e., blood, liver). In the blood, the levels of ApoE decreased in thepresence of probucol and continued to decrease with increasing doses.The levels of ApoE in the liver initially increased but then decreasedat the higher doses of probucol. At the highest administered dose (5mg), the levels of ApoE in the blood and liver were reduced to 60% ofthe control values for these tissues. In contrast, the levels of ApoE inthe brain increased to approximately 150% of the control value at thehighest dose evaluated. We believe the different responses mayreflect 1) differences in the distribution of probucol in the differenttissue compartments and/or 2) tissue specific effects of probucol on theexpression and secretion of ApoE. TABLE 3 In Vivo Effect of Probucol onApoE Levels in Different Murine Tissues* Probucol Dose G/DxA G/DensTissue Sample (mg) G/Dens-DD (% Total) (% Peak) Blood control 0.294120.274 74.900 0.1 0.2783 19.187 70.886 0.5 0.2262 14.885 57.610 5.00.1800 11.846 45.847 rApoE (10 ng) 0.0024 0.149 0.604 Hippocampuscontrol 0.0653 4.092 16.629 0.1 0.0729 4.570 18.571 0.5 0.0587 3.49714.958 5.0 0.1000 5.954 25.469 rApoE (20 ng) 0.0292 1.831 7.441 Livercontrol 0.0540 3.216 13.759 0.1 0.0840 5.263 21.386 0.5 0.0444 2.50611.315 5.0 0.0325 1.933 8.268

EXAMPLE 10 Human Clinical Trial to Determine the Effect of ProbucolAdministration on Cerebrospinal Fluid (CSF) ApoE Levels and on theClinical Manifestations of AD Patients with Different apoE Genotypes

[0129] We have conducted a clinical trial to investigate the potentialutility of probucol as a therapeutic agent for altering diseaseprogression in Alzheimer's disease. Our initial hypothesis was that ADpatients who are apoE4 carriers are characterized by a defective systemof repair due to lower ApoE brain levels. Accordingly, in thesepatients, it should be possible to either stop or slow down the diseaseprocess by increasing the amount of normally functioning ApoE.

[0130] To test this hypothesis, patients with AD with apoE3/3 and 4/3phenotypes were treated with probucol for a period of six months and theeffect of that treatment on the cerebrospinal fluid (CSF) levels of ApoEand on clinical scales of cognitive functioning were documented.

[0131] Experimental Design and Methods

[0132] A total of 8 patients with AD participated in this study. Afterdetermination of the ApoE phenotype at a baseline evaluation, allpatients received probucol.

[0133] Inclusion criteria were the following: 1) patients were between50 and 80 years of age; 2) women were post-menopausal or surgicallysterile; 3) patients had a clinical diagnosis of probable Alzheimer'sdisease; 4) subjects were fluent in French or English (able to write,read, speak, and understand); 5) the availability of an informant wasrequired; 6) patients had a severity index between 10 to 26 on theMini-Mental State Examination (MMSE); 7) patients who had taken aninvestigational drug less than four months before the baseline wereexcluded; and, 8) the following medications were disallowed (should notbe taken four months before baseline): cimetidine, lipophilicbeta-blocker (propranolol), clonidine, anticholinergics, andantidepressant with anticholinergic activity (tricyclics), neuroleptics,putative cognitive enhancers and central nervous system stimulants, orbenzodiazepines with a long half life.

[0134] The demographic data of the patients in this study are summarizedin Table 4. Patients with a clinical diagnosis of probable Alzheimer'sdisease were recruited through the memory clinic at the Douglas Hospital(Montreal, Canada). The mean age of onset of the disease was 65, youngerthan observed in most large clinical drug trials. These patients wereall in the early stages of the disease as measured by an average MMSEscore of 17.3, one year after their initial diagnosis. TABLE 4 PatientDemographic Data for Clinical Trial Patient Demographic Table age agePatient (study) Sex (diagnosed) education MMSE PP-101 58 2 57 11 15PP-102 66 1 64 10 19 PP-103 60 1 59 20 19 PP-104 78 1 77 7 20 PP-105 742 73 8 20 PP-106 62 1 62 12 20 PP-107 80 2 80 12 14 PP-108 61 1 61 7 16PP-109 56 1 52 9 13 Average 66.1 65 10.7 17.3

[0135] Primary Efficacy Parameters

[0136] Biological Marker: A lumbar puncture was performed at baselineand one month after treatment. The relative concentrations of ApoElevels in the CSF were determined by immunoblot as described by Poirer,J. et al., Neuroscience 55:81-90 (1993).

[0137] Neuropsychological Assessment: The Alzheimer's Disease AssessmentScale, Cognitive Part (ADAS-Cog) (see Rosen et al., Am. J. Psychiatr.,141:1356-1364 (1984)) was used as an objective neuropsychologicalmeasure. It consists of a battery of neuropsychological tests fordifferent cognitive functions. The battery comes to a single score witha maximum of 70 points and is administered in about 45 minutes topatients. A 117 point extended scale version of this test was also used(see Table 5).

[0138] Secondary Efficacy Parameters

[0139] Biological Markers: Additional biochemical markers includedphenotyping of the patients ApoE and determining relative concentrationsof tau protein, P-amyloid protein (1-42 and 1-40); and relative extentof lipid peroxidation.

[0140] Neuropsychological Assessment: The Clinical Impression of Change(CIBIC-Plus) and the Global Deterioration Scale (GDS) were recorded at 3and 6 months. The Clinical Impression of Change (CIBIC-Plus) consists ofthe holistic impression by the clinician of whether the patient hasshown any change since baseline as rated on a seven point sale (marked,moderate, mild improvement, no change, or marked, moderate, milddecline). The Global Deterioration Scale rates clinically identifiablestages of dementia.

[0141] Other tests that are useful for evaluating cognitive function inAD are the Disability Assessment in Dementia (DAD) questionnaire(Gauthier L et al., Abstract Book of the Sixth Congress of theInternational Psychogeriatric Assoc., 1993), the Cornell depressionscale (Alexopoulos et al. 1988)) and the Behave-AD test (Reisberg et al.1987). TABLE 5 Comparison of the Standard ADAS-Cog (70 point scoringmethod) and the Extended ADAS-Cog (117 point scoring method). Scale Item70 point scale 117 point scale word-recall task mean number of words notrecalled in 3 trials naming of objects and fingers 0 = 0-2 items namedincorrectly 1 = 3-5 2 = 6-8 3 = 9-11 4 = 12-14 5 = 15-17 commands pointsequal the number of points equal the incorrect commands (max = 5) numberof correct commands (max = 5) constructional praxis points equal thenumber of points for every incorrect drawings (max = 5) correct drawings(max = 5) ideational praxis points equal the number of points for eachcomponents failed (max = 5) successful components (max = 5) orientationpoints equal the number of points for each correct incorrect items (max= 8) item (max = 8) word-recognition task points equal the number ofpoints for each correct incorrectly recognized words identification (max= 12) (max = 48) spoken ability max 5 not included comprehension ofspoken max 5 not included language word finding difficulty in max 5 notincluded spontaneous speech remembering test instructions max 5 notincluded

[0142] Overview of Clinical Trial

[0143] Eight of the nine patients recruited for the study completed thesix month course of treatment. Non-compliance by one of the patients(patient PP-107) resulted in removal of the patient from the study afterthe three-month visit.

[0144] Adverse effects related to the administration of probucol areusually relatively minor (e.g., diarrhea, flatulence, abdominal pain,and nausea). Other occasional adverse effects include asymptomaticprolongation of the Q-T interval, eosinophilia, paresthesia, andangioneurotic edema (Buckley et al., Drugs, 37:761-800 (1989)). Noserious adverse events were reported. At the 3 month visit, theelectrocardiograms for two of the patients in the study group showed anasymptomatic prolongation of the QT interval. The electrocardiogramsnormalized after reducing the probucol dose from 1000 mg to 500 mg perday for the remaining three months of the study.

[0145] All nine patients underwent lumbar puncture scheduled at thefirst visit and after the first month. The CSF of 8 patients (1-6, 8,and 9) were analyzed for concentrations of ApoE and other biochemicalmarkers. The lumbar punctures were well tolerated with no reported sideeffects.

[0146] We observed in eight (8) patients receiving probucol at 1000mg/day, the concentration of ApoE in the cerebral spinal fluid (CSF)increased by an average of 125% (% of baseline) following one month oftreatment. Neuropsychological assessment of these patients (over the 6month course of probucol treatment) revealed a trend that suggested thatpatients with the greatest increase in CSF ApoE protein are more likelyto show an improvement in cognitive functions (as evaluated on theADAS-cog scale). These results establish the first etiologic treatmentof AD based on ApoE metabolism and support the theory linking apoE4 andAD.

[0147] Effect of probucol on the Concentration of, Apolipoprotein E inthe CSF

[0148] The cerebral spinal fluid (CSF) of the above subjects wasanalyzed for the concentration of ApoE protein by immunoblot using aninternal standard. The results of this analysis are summarized in Table6. The samples for the first six patients were analyzed independently ofthe last two patients, thus the scales for patients 1-6 are differentfrom the scales for patients 8 and 9. TABLE 6 Summary of ApoE Levels inCerebral Spinal Fluid ApoE Baseline ApoE 1 month Change in ApoE ApoEPatient (ROD) (ROD) (% baseline) Genotype PP-101 4.51 8.03 178% 3/3PP-102 10.87 14.81 136% 3/3 PP-103 7.71 10.27 133% 3/3 PP-104 2.20 2.59118% 4/3 PP-105 8.05 7.14  89% 3/3 PP-106 10.59 9.17  87% 4/3 PP-107PP-108 0.0713 0.1086 152% 4/3 PP-109 0.0819 0.0861 105% 4/3 AverageChange 125%

[0149] For the 8 patients receiving 1000 mg/day of probucol, theconcentration of ApoE in the CSF increased by an average of 125%following one month of treatment. These results are consistent with ourin vitro (Example 8) and in vivo animal results (Example 9). Twodifferent genotypes (phenotypes), the E3/E3 and E4/E3, were representedin this group of patients. On average, the E3/E3 group appeared torespond better than the E4/E3 group, however the small study grouplimits any interpretation of genotype influence on ApoE induction.

[0150] Neuropsychological Assessments

[0151] In order to determine if probucol-induced increases in ApoElevels influenced patient outcome we conducted ADAS-Cog tests (70 point,and extended 117 point) to measure any changes in the cognitivefunctioning in patients with Alzheimer's disease. The range ofparameters measured in each of the ADAS-Cog tests is summarized in Table5. These tests are used as the standard instrument for demonstratingcognitive efficacy in Alzheimer's Disease drug trials, particularly inNorth America. The ADAS includes both cognitive (ADAS-Cog) andnon-cognitive (ADAS-Noncog) subscales and the tests components ofmemory, language, and behavior. The standard ADAS-Cog test (70 pt.) isscored by errors, and a higher score indicates poorer performance.

[0152] Cognitive Function in AD Patients after Treatment with Probucol

[0153] We assessed the cognitive function of all subjects who completed6 months of probucol treatment using the ADAS-Cog test (70 pt. scale).As shown in Table 7 and graphically in FIG. 9, a general trend in thedata suggests that patients with the greatest increases in the apparentlevels of ApoE protein in the CSF (measured at one month), are morelikely to be stabilized or improved as judged by the changes in theADAS-Cog scores over the 6 month course of treatment. We furtherassessed the cognitive functioning of these patients with the extendedscale ADAS-Cog test. TABLE 7 ADAS-Cog (70 point scale) Scores forPatients that Completed Six Month Treatment with Probucol ADAS-Cog (70point scale) Patient Baseline 3 months 6 months PP-101 31 29 25 PP-10234 34 36 PP-103 24 28 23 PP-104 30 37 40 PP-105 41 41 44 PP-106 20 22 16PP-107 withdrawn PP-108 19 18 17 PP-109 32 27 40 Average 28.9 29.5 30.13

[0154] The extended 117 pt. ADAS-Cog test represents an alternativescoring approach that places more emphasis on the verbal memorydemonstrated by the patient. The scoring method for this modifiedADAS-Cog test ranges from 0 (the lowest score) to 117 (the best possiblescore).

[0155] As summarized in Table 8, five of the patients appeared tostabilize or improve over the course of the six month treatment. Inparticular, three of these patients showed significant increases (7-9points). The remaining three patients decreased relative to baselinescores. TABLE 8 Patient Cognitive Performance (as measured by ADAS-Cog(extended 117 point scale) during the Six Month Course of Treatment withProbucol ADAS-Cog (117 point scale) Patient Baseline 3 months 6 monthsGenotype PP-101 64 63 72 E3/E3 PP-102 64 63 65 E3/E3 PP-103 72 73 74E3/E3 PP-104 56 51 49 E4/E3 PP-105 73 73 82 E3/E3 PP-106 66 63 63 E4/E3PP-107 withdrawn PP-108 75 83 82 E4/E3 PP-109 64 56 58 E4/E3 Average66.8 65.6 68.1

[0156] The initial hypothesis of this study was that probucol wouldexert a beneficial effect on the progression of the disease byincreasing the ApoE levels in the brain. FIGS. 10 and 11 summarize thechanges in the ADAS-Cog scores (117 pt. extended scale) as a function ofthe change in ApoE protein level in the CSF.

[0157] At 3 and 6 months, there is no significant correlation betweenthe change in ADAS-cog scores (117 pt. extended scale) and the change inApoE protein levels in the CSF. However, if patient PP-105 is consideredan outlier, a correlation (p<0.02) is observed at six months indicatingthat a higher ApoE level results in higher cognitive performance.

[0158] CIBIC-Plus

[0159] Using the Clinical Impression of Change (CIBIC-Plus) we furtheranalyzed the effects of probucol on the cognitive function of ADpatients. This test provides a holistic impression by the clinician ofwhether the patient has shown any change since baseline. The CIBIC isscored as a seven point scale, with a range from 1=“very much improved”to 4=“no change” to 7=“very much worse.” Changes in the CIBIC score haveshown only weak correlation with changes in the ADAS-Cog and have beenreported to be less sensitive to change than other measures. The FDAguidelines are widely interpreted as mandating the use of the CIBIC andthis scale serves to measure the impact of treatment protocols on thequality of life for the patient.

[0160] As summarized in Table 9, the scores for the CIBIC-Plusevaluation indicate that there was a slight deterioration during the 6month course of treatment for the group as a whole. TABLE 9 Effect ofProbucol Treatment on the Overall Impression of Change (Measured byCIBIC Plus) CIBIC Scores Patient 3 months 6 months PP-101 4 5 PP-102 5 5PP-103 4 6 PP-104 3 5 PP-105 5 5 PP-106 3 6 PP-107 4 Withdrawn PP-108 44 PP-109 3 6 Average 3.9 5.3

[0161] Global Deterioration Scale

[0162] To further extend our studies on the effect of probucol on ADpatient outcome, we employed the Global Deterioration Scale (GDS). TheGDS test provides a description of the key features of AD progression.The GDS rates clinically identifiable stages (i.e., no cognitivedecline; subjective complaints without an objective deficit; subjectivecomplaints with some objective deficits which do not meet dementiacriteria; mild dementia; moderate dementia; severe dementia; andterminal dementia). The GDS is calculated as the average of scoresrelated to the performance in the following areas of attention andconcentration, recent memory, past memory, orientation, and functioningand self care.

[0163] The results of this study as shown in Table 10, indicate thatthere was a relative stabilization in patient symptoms at three and sixmonths. TABLE 10 Results of the Global Deterioration Scale at 3 and 6Months Global Deterioration Scale Patient Baseline 3 months 6 monthsPP-101 3.5 1.5 1 PP-102 5 8 8 PP-103 3 3.25 3.5 PP-104 3.5 3.5 5.5PP-105 5.5 5 6.5 PP-106 5.5 7 7 PP-107 5.5 5.5 withdrawn PP-108 6 4.5 5PP-109 5 5 6 Average 4.7 4.8 5.3

[0164] Secondary Biochemical Markers

[0165] β-Amyloid Protein

[0166] The effects of probucol on the CSF levels of total β-amyloidprotein (1-40) and (1-42) were determined after one month of treatmentwith probucol. Tables 11 and 12 summarize the changes in total β-amyloidprotein (1-40 and 1-42) at one month, as a percentage of the baselinevalues.

[0167] The results indicate an overall increase in the levels ofβ-amyloid in the CSF of these Alzheimer's patients treated withprobucol. In addition, the increase in total β-amyloid appeared to beselective for the E3/E3 genotype (Table 11). These increases in CSFβ-amyloid are interesting in view of the fact that it has been reportedthat CSF amyloid concentration (particularly β-amyloid 1-42) decreasesas the severity of the dementia increases. TABLE 11 Changes in the CSFConcentrations of Total B-amyloid (1-40) Following One Month ofTreatment with Probucol [β amyloid 1-40] in the CSF (average oftriplicate assays) Patient Baseline 1 month % change Genotype PP-1010.546 5.35 979 E3/E3 PP-102 0.100 2.16 2157 E3/E3 PP-103 0.996 3.44 345E3/E3 PP-104 3.67 3.95 108 E4/E3 PP-105 4.96 4.96 100 E3/E3 PP-106 3.137.55 241 E4/E3 PP-107 PP-108 3.10 3.26 105 E4/E3 PP-109 3.27 3.51 107E4/E3

[0168] TABLE 12 Changes in the CSF Concentrations of β-amyloid (1-42)Following One Month of Treatment with Probucol [β amyloid 1-42] in theCSF (average of triplicate assays) Patient Baseline 1 month % changePP-101 0.17 1.06 624 PP-102 0.06 1.29 2144  PP-103 1.02 2.48 242 PP-1042.76 3.92 142 PP-105 1.35 0.76  56 PP-106 1.25 1.78 142 PP-107 PP-1081.10 2.15 195 PP-109 ND 0.31 —

[0169] Tau Protein

[0170] We summarize in Table 13 the change in Tau protein concentrationin the CSF at one month relative to baseline. We observed that, as agroup, there was no effect of probucol on Tau levels in this study groupof Alzheimer's patients. Although probucol treatment affected the levelsof ApoE and beta-amyloid in the CSF, the absence of an effect on Taulevels demonstrates the selectivity of the probucol effects. TABLE 13Levels of Tau Protein in the CSF at Baseline and After One Month ofTreatment with Probucol Tau concentration in the CSF Patient Baseline 1month % change PP-101 420 394 94 PP-102 700 916 131 PP-103 627 620 99PP-104 201 188 93 PP-105 1012 1099 109 PP-106 200 251 97 PP-107 11621070 92 PP-108 448 458 102 PP-109 993 1120 113

EXAMPLE 11

[0171] Analysis of the Anti-Oxidant Properties of Probucol

[0172] In order to assess the antioxidant properties of probucol weanalyzed lipid peroxidation levels in the CSF of patients before andafter one month of treatment with probucol (one month). In vitro lipidperoxidation was measured according to the method of Ohkawa et al.(Anal. Biochem., 95:351-358 (1979)) with some modifications.

[0173] Briefly, cerebrospinal fluid samples from Alzheimer's diseasepatients were prepared in 20 volumes of phosphate buffer (pH=7) (95% N₂,5% CO₂) with a Teflon homogenizer. After centrifugation (1000 g, 10min., 2° C.), the supernatant was precipitated with trichloroacetic acid(72%) according to Brown et al. (Anal. Biochem., 189:136-139 (1989)).This procedure removed material such as non-lipid-derivedmalondialdehyde (MDA) that could interfere with lipid peroxidationmeasurement. After precipitation with trichloracetic acid andcentrifugation (10000 g, 15 min., 2° C.), 100 μl of the supernatantcontaining human brain homogenates was used to measure MDA levels. Thesesupernatants were incubated with 20 μl of SDS 10%, 150 μl of acetic acid20% (pH 3.5), and 150 μl of thiobarbituric acid (TBA) (0.6% in 0.05 NNaOH) and heated at 100° C. for 60 min. This step was performed under acontrolled atmosphere (95% N², 5% CO²) in sealed vials to avoidformation of MDA during heating. At the end of the incubation, thereaction was stopped at 4° C. The MDA-TBA complex of TBARS(thiobarbituric acid reactive substances) was then extracted with 300 μlof butanol/pyridine (15:1) by strong shaking (for 30 sec.) followed bycentrifugation (1000 g, 10 min., 4° C.). TBARS present in the organicphase was than measured in a Perkin-Elmer spectrofluorophotometer(connected to a microplate reader (96 wells) with λ excitation of 532 nmand λ emission at 553 nm) and results are reported in Table 14. TABLE 14Change in Lipid Peroxidation After Treatment with Probucol LipidPeroxidation (Fluorescence Units) Patient baseline 1 month Change (%baseline) PP1-101 0.745 0.691 92.8 PP1-102 0.704 0.731 103.8 PP1-1030.710 0.824 116.1 PP1-104 0.703 0.705 100.3 PP1-105 0.704 0.686 97.4PP1-106 0.725 0.687 94.8 PP1-107 PP1-108 0.710 0.692 97.5 PP1-109 0.7140.744 104.2 Change (% Baseline) 100.9

[0174] After 1 month of dosing with probucol, the average for eightpatients indicated no changes in the levels of lipid peroxidation in theCSF relative to baseline values. For individual patients, any changes inlipid peroxidation (higher or lower) were small.

[0175] In addition to the relationship between probucol and lipidperoxidation, we wanted to determine if the antioxidant properties ofprobucol had any effect on ApoE levels and AD patient outcome. Weperformed a linear regression analysis curve that examined therelationship between the change in lipid peroxidation (% baseline) as afunction of a change in ApoE levels (FIG. 12). We also performed asimilar analysis that examined the relationship between the change inADAS-Cog scores (70 pt. and 117 pt.; six months-baseline) as a functionof any change in lipid peroxidation levels (FIGS. 13 and 14).

[0176] As shown in FIG. 12, we observed no correlation between changesin lipid peroxidation and ApoE induction. Similarly, as shown in FIGS.13 and 14, no correlation between changes in individual ADAS-Cog scores(70 pt or 117 pt) and changes in lipid peroxidation were observed. Thus,although our study group was small, we concluded that probucol exertsits pharmacologic effect primarily through mechanisms related to ApoEinduction.

EXAMPLE 12

[0177] Animal Models for Determining the Efficacy of Probucol inTreating Stroke

[0178] To demonstrate if increasing ApoE levels with probucol provides aneuroprotective effect in treating stroke, several animal models knownin the art may be employed (e.g., Simpkins et al., J. Neurosurg.87(5):724-730 (1997); Nawashiro et al., J. Cereb. Blood Flow Metab.17(5)483-490 (1997); and Connolly et al., Neurosurgery 38(3):523-531(1996)).

[0179] In particular, the Wistar rat model may be used withmodifications. Briefly, rats are dosed (i.p.) daily with probucol or asaline negative control for ten days. On the tenth day, animals are thensubjected to a 2 hour reversible, middle cerebral artery occlusion.Laser-Doppler flowmetry is used to monitor the ischemic period and earlyreperfusion. After 22 hours of reperfusion, the infarct volume is thendetermined by 0-2, 3, 5-triphenyltetrazolium chloride staining and imageanalysis.

[0180] We predict that the cortical infarct volumes (indicative ofneuronal damage) will be less in animals treated with probucol ascompared to untreated animals.

[0181] To extend these animal studies, the ApoE knockout mouse may beexploited to assess the role of ApoE in probucol-mediatedneuroprotection for stroke. This model will permit the evaluation ofprobucol in animals where ApoE can be induced (wild type) or isnon-existent due to an apoE gene disruption.

[0182] This protocol is carried out as similarly described for the ratmodel above, with some modifications. Briefly, mice (wild type orApoE-knockout) are dosed (i.p.) daily with probucol (5 mg) or a salinenegative control for ten days. On the tenth day, animals are subjectedto a transient focal ischemia by intraluminal occlusion of the middlecerebral artery. Infarct volume, neurological outcome, and cerebral flowwill be monitored. Our prediction is that treatment with probucol wouldbe expected to have little or no benefit in the knockout animals if ApoEinduction is necessary for the neuroprotective properties of probucol intreating stroke.

[0183] It will be appreciated that these protocols may be modified toalso determine the effect of probucol in treating sudden acute stroke inan animal (probucol administered after the induced ischemic incident).In addition, the above stroke model may be employed using larger mammals(e.g., cats, dogs) for formulating preclinical dosage ranges, methods ofadministration, or other parameters in preparation for carrying out ahuman clinical trial.

Other Embodiments

[0184] Preferably, compositions containing probucol (Lorelco™,manufactured by Marion Merrell Dow Inc.), tacrine (Cognex™, manufacturedby Parke-Davis), heptylphysostigmine (manufactured by Merck Corp.),Simvastatin (Zocor™, manufactured by Merck Corp.), lovastatin (Mevacor™,manufactured by Merck Corp.), pravastatin (Pravachol™, manufactured byBristol-Myers Squibb), thorvastatin (Parke-Davis), probucol analogs,vitamin E, donepezil (Aricept™, marketed by Pfizer), blood pressureinhibitors, antioxidants, anti-inflammatories, and steroids are suitablefor altering ApoE as described herein and useful for the treatment ofhuman patients, but these compounds may also be used to treat any othermammal, for example, any pet or domesticated livestock. Anyneurodegenerative problems associated with the types of altered brainfunction described herein may be improved with any of the compositionsdescribed above.

[0185] In addition, normal nervous system biology may also be enhancedby the administration of compositions described herein with improvementsin cognitive performance or other nervous system performance being theresult.

[0186] For any of these additional uses, these compositions as describedabove are administered by the general methods described herein.

[0187] All publications and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

[0188] While the invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure come within known or customary practice within theart to which the invention pertains and may be applied to the essentialfeatures hereinbefore set forth, and follows in the scope of theappended claims.

[0189] Other embodiments are within the claims.

What is claimed is:
 1. A method of treating a neuronal deficit in a patient, said patient diagnosed with a neurodegenerative disease or a predisposition to a neurodegenerative disease, said method comprising administering a therapeutically-effective amount of a composition other than tacrine, donepezil, or estrogen, said composition being a composition which increases ApoE levels.
 2. The method of claim 1 , wherein, said neurodegenerative disease is a disease other than AD.
 3. The method of claim 1 , wherein said neurodegenerative disease is a disease other than stroke.
 4. The method of claim 1 , wherein said composition comprises a compound selected from the group comprising probucol, heptylphysostigmine, simvastatin, lovastatin, pravastatin, and thorvastatin.
 5. A method of treating a neuronal deficit in a patient, said patient diagnosed with a non-Alzheimer's neurodegenerative disease or a predisposition to a non-Alzheimer's neurodegenerative disease, said method comprising administering a therapeutically effective amount of a composition which increases ApoE levels.
 6. The method of claim 5 , wherein said composition comprises a compound selected from the group comprising probucol, tacrine, heptylphysostigmine, simvastatin, lovastatin, pravastatin, thorvastatin, and steroids.
 7. The method of claim 5 , wherein said composition comprises estrogen.
 8. The method of claim 1 , wherein said method includes administering a second compound, said second compound selected from the group comprising probucol analogs, vitamin E, donepezil, blood pressure inhibitors, antioxidants, anti-inflammatories, or steroids other than estrogen.
 9. The method of claim 5 , wherein said method includes administering a second compound, said second compound selected from the group comprising probucol analogs, tacrine, vitamin E, donepezil, blood pressure inhibitors, antioxidants, anti-inflammatories, or steroids.
 10. The method of claims 1 or 5, wherein said amount of a composition is sufficient to increase ApoE levels in said patient.
 11. The method of claim 10 , wherein said amount of a composition is sufficient to increase ApoE levels by about 10% or more.
 12. The method of claims 1 or 5, wherein said amount of a composition is sufficient to increase amyloid scavenging by ApoE.
 13. The method of claims 1 or 5, wherein said patient has a disease of the central nervous system.
 14. The method of claims 1 or 5, wherein said patient has a disease of the peripheral nervous system.
 15. The method of claims 1 or 5, wherein said patient is presymptomatic.
 16. The method of claims 1 or 5, wherein said patient has at least one apoE4 allele.
 17. The method of claims 1 or 5, wherein said patient has been diagnosed to suffer from, Creutzfeldt-Jakob disease, Huntington's disease, Lewy body disease, Parkinson's disease, Pick's disease, amyotrophic lateral sclerosis, neurofibromatosis, brain injury, stroke, multiple sclerosis, or multiple infarct dementia.
 18. The method of claim 1 , wherein said patient has been diagnosed to suffer from Alzheimer's disease.
 19. The method of claims 1 or 5, wherein said patient is genetically predisposed to suffer from a disease selected from the group consisting of Creutzfeldt-Jakob disease, Huntington's disease, Lewy body disease, Parkinson's disease, Pick's disease, amyotrophic lateral sclerosis, multiple sclerosis, stroke, multiple infarct dementia, and neurofibromatosis.
 20. The method of claim 1 , wherein said patient is genetically predisposed to suffer from Alzheimer's disease.
 21. The method of claim 1 , wherein said administering further includes administering tacrine to said patient.
 22. The method of claim 1 , wherein said administering further includes administering estrogen to said patient.
 23. The method of claim 1 , wherein said administering further includes administering donepezil to said patient.
 24. A method of increasing ApoE polypeptide levels in a patient, said method comprising administering a therapeutically-effective amount of a composition comprising probucol or an analog of probucol.
 25. The method of claim 24 , wherein said patient has a neurodegenerative disease.
 26. The method of claim 24 , wherein said patient has a disease of the central nervous system.
 27. The method of claim 24 , wherein said patient has a disease of the peripheral nervous system.
 28. The method of claim 24 , wherein said patient is suffering from Alzheimer's disease.
 29. The method of claim 24 , wherein said patient is genetically predisposed to suffer from Alzheimer's disease.
 30. The method of claim 24 , wherein said patient has at least one apoE4 allele.
 31. The method of claim 24 , wherein said patient is suffering from Creutzfeldt-Jakob disease, Huntington's disease, Lewy body disease, Parkinson's disease, Pick's disease, amyotrophic lateral sclerosis, neurofibromatosis, brain injury, stroke, multiple sclerosis, or multiple infarct dementia.
 32. The method of claim 24 , wherein said patient is genetically predisposed to suffer from a disease selected from the group consisting of Creutzfeldt-Jakob disease, Huntington's disease, Lewy body disease, Parkinson's disease, Pick's disease, amyotrophic lateral sclerosis, multiple sclerosis, stroke, multiple infarct dementia, and neurofibromatosis.
 33. The method of claim 24 , wherein said therapeutically effective amount of a composition may comprise a compound selected from the group comprising probucol analogs, tacrine, heptylphysostigmine, simvastatin, lovastatin, pravastatin, thorvastatin, vitamin E, donepezil, blood pressure inhibitors, antioxidants, anti-inflammatories, and steroids.
 34. The method of claim 24 , wherein said composition further includes one or more compounds selected from the group comprising, vitamin E, donepezil, blood pressure inhibitors, antioxidants, anti-inflammatories, and steroids.
 35. The method of claim 24 , wherein said expression is measured by quantitating ApoE polypeptide levels.
 36. The method of claim 24 , wherein said expression is measured by quantitating ApoE polypeptide levels with an antibody.
 37. The method of claim 24 , wherein said increasing is in brain tissue or human cerebrospinal fluid of said patient.
 38. A method of identifying a therapeutic compound effective for the treatment of a neurodegenerative disease, said method comprising exposing a cell to a compound and measuring the expression of ApoE, an increase in said expression identifying a therapeutic compound.
 39. The method of claim 38 , wherein an increase ApoE levels by 10% or more indicates a therapeutic compound.
 40. The method of claim 38 , wherein said cell is an astrocyte, neuron, glial cell, oligodendrocyte, Schwann cell or any other cell derived from nervous system tissue.
 41. The method of claim 38 , wherein said cell is derived from nervous system tissue from a patient with, or predicted to suffer from, a neurodegenerative disease.
 42. The method of claim 38 , wherein said expression is measured by quantitating apoE mRNA levels.
 43. The method of claim 38 , wherein said expression is measured by quantitating ApoE polypeptide levels. 